Dog PrP sequence alignments.

<p><b>A</b>. PrP amino acid alignment based on residues 91–230 (dog PrP numbering) from species with different experimental and/or natural susceptibilities to prion diseases. Classification criteria were based on the number of representative prion strains able to be transmitted and cause disease in the host. Identical amino acids are indicated by dots. Δ: number of different amino acids compared to dog PrP. Note the similarity of the amino acid sequence in the cat (highlighted in red), a species known to be susceptible to naturally acquired prion disease. <b>B</b>. Upper line, dog amino acid residues 91–230. The 6 amino acid differences compared to cat PrP sequence are highlighted by red squares. Boxes show each highlighted amino acid and representative species in which the particular amino acid is present also. Where prion susceptibility of the species has been proven the reference is provided. NP: susceptibility not proven. Positions 101 and 163 are polymorphic in dogs (Ser/Gly and Asp/Glu, respectively). Species codification and accession numbers: doPrP, dog PrP (FJ870767.1); hoPrP, horse PrP (ACG59277); raPrP, rabbit PrP (NP001075490); poPrP, porcine PrP (AAA92862.1); caPrP, cat PrP (ACB97675.1); huPrP, human PrP (NP001073592); mdPrP, mule deer PrP (AY330343.1); moPrP, mouse PrP (NP035300); boPrP, bovine PrP (ABR92636.1); ovPrP, ovine PrP (NP001009481.1); bvPrP, bank vole PrP (AAL57231.1); ahPrP, armenian hamster PrP (AAA37014); rcPrP, raccoon PrP (ACA50738.1); rsPrP, red squirrel PrP (AAN16491); coPrP, coyote PrP (AGA63673); gwPrP, grey wolf PrP (AGA63687); rfPrP, red fox PrP (ACA50742); huPrP; human PrP (U29185.1); niPrP, nilgai PrP (AAV30507); baPrP, California big-eared bat (AAN16503); anPrP, anteater PrP (AAN16516).</p

Unraveling the key to the resistance of canids to prion diseases

<div><p>One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different <i>in vitro</i> and <i>in vivo</i> models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the <i>Leporidae</i> and <i>Equidae</i> families. Here we undertake <i>in vitro</i> and <i>in vivo</i> approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis <i>in silico</i>, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.</p></div

<i>In vitro</i> propagation experiments.

<p><b>A & B</b>. Rounds (R1-R10) of serial PMCA using brain homogenates from two different breeds of dog as substrates: Cocker Spaniel and German Wirehaired Pointer. Several procedures to propagate different inocula over dog species were tested. <i>A1</i>: Standard 1–10% seeded PMCA with serial 1:10 dilution rounds. <i>B1</i>: 30–50% seeded PMCA with serial 1:10 dilution rounds. <i>B2</i>: same as B1 with the addition of zirconia-silica beads [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006716#ppat.1006716.ref052" target="_blank">52</a>]. <i>C1</i>: 10% seeded PMCA with serial 1:2 dilution rounds. <i>D1</i>: 30–50% seeded PMCA with serial 1:2 dilution rounds. Different inocula or mix of inocula were used as seed. <i>Mouse strain pool</i>: Pool of mouse PrP^Sc strains containing equal amounts of ME7, RML, 22F, 22L, 87V, 22A, 79A, 139A and other spontaneously <i>in vitro</i> obtained strains. <i>Sheep strain pool</i>: Pool of sheep PrP^Sc strains containing equal amounts of 7 different scrapie isolates. <i>Cervid strain pool</i>: Pool of cervid PrP^Sc strains containing equal amounts of 2 mule deer and 2 elk isolates. <i>BSE-C</i>: Three different isolates of classical BSE (isolate 1, isolate 2 and isolate 3). <i>BSE-L</i>: Atypical L-type BSE. <i>sBSE</i>: Sheep BSE. * Twelve unseeded tubes were extended to 20 rounds for each set of experiments. Dog (Cocker Spaniel)-BSE obtained after the propagation of BSE-C (isolate 3) was shown in Vidal <i>et al</i>. 2013 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006716#ppat.1006716.ref042" target="_blank">42</a>]. <b>C</b>. One tube of round 10 of each PK-resistant sample was selected to show the biochemical analysis of BSE-C (1; isolate 1 and 2; isolate 2) and sheep BSE (sBSE) seeded material generated by PMCA compared to brain-derived BSE-C and RML. Samples were digested with 85 μg/ml PK and analyzed by Western blot using monoclonal antibody D18 (1:5,000). PK: Protease-K. Mo: Mouse. Co: Cow. Do: Dog. Dog brain: negative control; undigested dog whole brain homogenate. Mw: Molecular weight.</p

Electrostatic potentials on the surface of protein structures.

<p><b>A.</b> Wild-type mouse prion protein (PDB ID: 4MA7). <b>B.</b> Canine prion protein (PDB ID: 1XYK). <b>C-E.</b> Modelled structures: Model01 <b>(C)</b>, Model02 <b>(D)</b>, and distribution of positively charged residues surrounding Asp158 in wild-type canine prion protein <b>(E)</b>. Acidic regions are colored in red and basic regions in blue, residue 158 for all structures is highlighted in dashed line white box. Amino acid D158 located within an area with four Arg/Lys residues (R135, R150, R155 and K119) introduces a positively charged residue in that region, in dog PrP, Model01 and Model02 changing the local charge distribution compared to mouse wild-type PrP.</p

Inoculation of mouse-derived prion strains into Tg402, Tg403 and C57BL/6 mice.

<p>Inoculation of mouse-derived prion strains into Tg402, Tg403 and C57BL/6 mice.</p

<i>In vitro</i> propagation ability of RML, 301C and 22L mouse prion strains by PMCA.

<p>10% brain homogenates from transgenic N158D PrP mouse lines (Tg402 and Tg403) were seeded with the different mouse prion strains (RML, 301C and 22L) at the indicated dilutions and subjected to one single 48h PMCA round. All samples [non-PMCA amplified (NA) and amplified samples (A)] were digested with 85 μg/ml of protease-K (PK) and were analyzed by Western blot using SAF-83 (1:400) monoclonal antibody. No mouse prion strains were able to propagate in any of the transgenic N158D PrP mouse brain homogenates. C(Mo): undigested mouse brain homogenate. Mw: Molecular weight.</p

Two cell/brain-PMCA methodologies to study the effect of the residue 158 in mouse PrP.

<p><b>A</b>. Schematic representation of the cell/brain-PMCA propagation study. Brain-derived PrP^C (black filled circles) is mixed with cell-derived 3F4-tagged PrP^C (grey filled circles) and seeded with PrP^Sc (RML or 22L, black filled squares). The resulting 3F4-tagged PrP^Sc (grey filled squares) is specifically detected by the 3F4 antibody. <b>B</b>. A 1:40 dilution of RML or 22L were used as seeds for a PMCA based on mouse brain homogenate mixed with cellular 3F4-tagged substrates containing mouse PrP N158, D158, E158 or without PrP (<i>PRNP</i>^<i>0/0</i>; KO-PrP). Non-PMCA amplified samples and samples subjected to one 24 h single round of PMCA were digested with PK (20 μg/ml) and analyzed by Western blot using monoclonal antibody 3F4 (1:10,000). Control: undigested human brain homogenate. <b>C</b>. Schematic representation of the cell/brain-PMCA inhibition studies. Brain derived PrP^C (black filled circles) is mixed with cell derived PrP^C (black filled circles) and seeded with PrP^Sc (RML, black filled squares). Total resulting PrP^Sc is detected by antibody D18. <b>D</b>. 1:5,000 or 1:10,000 dilutions of RML were used as seeds for a PMCA based on mouse brain homogenate mixed with cellular substrates containing mouse PrP N158, D158, E158 or without PrP KO-PrP. Samples subjected to one 24 h single round of PMCA were digested with PK (20 μg/ml) and detected by the monoclonal antibody D18 (1:10,000). Both procedures showed a significant inhibitory effect of the D158 and E158 substitutions over the <i>in vitro</i> propagation of RML/22L mouse prion strains. Control: undigested mouse brain homogenate. Mw: Molecular weight.</p

Brain analyses from RML, 301C and 22L inoculated mice.

<p><b>A</b>. Two mouse brains from each inoculated group (Tg402, Tg403 and C57BL/6) were selected to determine the presence of protease-K (PK) resistant PrP. Samples were treated with 85 μg/ml of PK and protease resistant protein was analyzed by Western blot using SAF-83 (1:400) monoclonal antibody. Only C57BL/6 inoculated brain samples showed characteristic PK-resistant PrP migration patterns. All transgenic mouse brains were devoid of all PK-resistant PrP. C(Mo): undigested mouse brain homogenate. <b>B</b>. Histopathological characterization of different mouse prion strains (RML, 301C and 22L) inoculated intracerebrally in Tg402, Tg403 and C57BL/6 wild-type mice. Two mouse-adapted scrapie derived strains (RML and 22L) and a mouse-adapted BSE derived strain (301C) showed typical spongiform change only in the brains of wild-type mice in hematoxylin and eosin (H&E) stained sections. No TSE-related lesions were observed in Tg402 nor in Tg403 inoculated mice with any of the strains. Similarly, upon immunohistochemical labeling with 6H4 antibody against prion protein [PrP^res IHC (6H4)] (1:1,000), only the wild-type mice had immunolabeled deposits of prion protein. All images were taken at the same magnification in the region of the diencephalon. WT: Wild-type. Bar 50 μm.</p

An antipsychotic drug exerts anti-prion effects by altering the localization of the cellular prion protein

<div><p>Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrP^C), an endogenous membrane glycoprotein of uncertain function, into PrP^Sc, a pathological isoform that replicates by imposing its abnormal folding onto PrP^C molecules. A great deal of evidence supports the notion that PrP^C plays at least two roles in prion diseases, by acting as a substrate for PrP^Sc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrP^C may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrP^C may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrP^C. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrP^C at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrP^C from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrP^C, as well as in the propagation of infectious prions.</p></div

CPZ alters the cell surface localization of PrP^C.

<p><b>A.</b> Cells were seeded on glass coverslips and grown for 24 h to ~60% confluence. For surface staining of PrP, cells were first incubated at 4°C with antibody D18 diluted, then fixed with paraformaldehyde and incubated with fluorescently-labelled secondary antibody. For total PrP staining, cells were permeabilized with Triton X-100, fixed with paraformaldehyde, and then incubated with primary and secondary antibodies. Coverslips were mounted with Fluor-save Reagent (Calbiochem), and analyzed with a Zeiss Imager M2 microscope. <b>B.</b> N2a cells stably expressing mouse WT PrP^C were grown to confluence on glass coverslips, and treated with the indicated concentrations of Fe(III)-TMPyP or CPZ for 24h. For detection of surface PrP^C (SC#1, shown in the picture), coverslips were incubated in ice with antibody 6D11 (this step was omitted for detection of total PrP^C, not shown). Coverslips were blotted on a nitrocellulose membrane soaked in lysis buffer, and incubated with horseradish peroxidase-conjugated secondary antibody. For detection of total PrP^C, cell blots were incubated with the primary and secondary antibodies. The PrP^C signal was revealed by enhanced chemiluminescence. <b>C.</b> PrP^C signal was quantitated by densitometry. The bar graph shows the % ratio of surface to total PrP^C. Each bar represents the mean (± standard error) of three independent experiments (n = 3). Statistically-significant differences (*), estimated by Student <i>t</i>-test, between CPZ-treated and untreated cells were as follow: [3 μM], <i>p</i> = 0.0058; [10 μM], <i>p</i> = 0.00034.</p